Protective Effects of Compound FLZ, a Novel Synthetic Analogue of Squamosamide, on Β-Amyloid-Induced Rat Brain Mitochondrial Dysfunction in Vitro

npg Acta Pharmacol Sin 2009 May; 30 (5): 522–529

Original Article

Protective effects of compound FLZ, a novel synthetic analogue of squamosamide, on β-amyloid-induced rat brain mitochondrial dysfunction in vitro

Fang FANG§, Geng-tao LIU*

Department of Pharmacology, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China

Aim: The aim of the present study was to assess the effects of N-[2-(4-hydroxyphenyl)ethyl]-2-(2,5-dimethoxyphenyl)-3-(3- methoxy-4-hydroxyphenyl) acrylamide (compound FLZ), a novel synthetic analogue of squamosamide, on the dysfunction of rat brain mitochondria induced by Aβ25–35 in vitro.

Methods: Isolated rat brain mitochondria were incubated with aged Aβ25–35 for 30 min in the presence and absence of FLZ

(1−100 μmol/L). The activities of key mitochondrial enzymes, the production of hydrogen peroxide (H2O2) and superoxide ·- anion (O2 ), and the levels of glutathione (GSH) in mitochondria were examined. Mitochondrial swelling and the release of cytochrome c from mitochondria were assessed by biochemical and Western blot methods, respectively.

Results: Incubation of mitochondria with aged Aβ25–35 inhibited the activities of α-ketoglutarate dehydrogenase (α-KGDH), ·- pyruvate dehydrogenase (PDH) and respiratory chain complex IV. It also resulted in increased H2O2 and O2 production, and decreased the GSH level in mitochondria. Furthermore, it induced mitochondrial swelling and cytochrome c release from the mitochondria. The addition of FLZ (100 μmol/L) prior to treatment with Aβ25–35 significantly prevented these toxic effects of Aβ25–35 on the mitochondria.

Conclusion: FLZ has a protective effect against Aβ25–35-induced mitochondrial dysfunction in vitro.

Keywords: compound FLZ; β-amyloid; mitochondrial dysfunction; mitochondrial key enzymes; cytochrome c Acta Pharmacologica Sinica (2009) 30: 522–529; doi: 10.1038/aps.2009.45

Introduction against AD[1, 11]. The natural product squamosamide was isolated from Mitochondria play an important role in the regulation of the leaves of Annona squamosa. Compound FLZ (N-[2- cell survival and death. Many lines of evidence suggest that (4-hydroxyphenyl)ethyl]-2-(2,5-dimethoxyphenyl)-3-(3- mitochondrial dysfunction is critical to the pathogenesis methoxy-4-hydroxyphenyl) acrylamide) is a novel synthetic of Alzheimer’s disease (AD) and other neurodegenerative [1–4] cyclic derivative of squamosamide with a molecular weight disorders . Additionally, mitochondrial dysfunction has of 449.5 (Figure 1). Our previous studies demonstrated that been proposed as a hallmark of β-amyloid (Aβ)-induced compound FLZ protected against dopamine- and MPP+-in- neuronal toxicity in the development and pathogenesis of [5–10] duced apoptosis in PC12 and SH-SY5Y cells. This protective AD . Therefore, anti-AD pharmacological studies have effect is thought to be the result of inhibition of cytochrome focused intensively on the potential protective effects of c release from mitochondria and subsequent caspase-3 stabilizing or restoring mitochondrial function as a therapy activation[12, 13]. In addition, our in vivo studies also showed that compound FLZ improved abnormal behaviors caused * Correspondence to Prof Geng-tao LIU. by the functional disturbance of dopaminergic and cholin- E-mail [email protected]/[email protected] ergic neurons. Furthermore, this compound also improved § Now at the Department of Pharmacology, School of Chinese Materia the prognosis of mouse Parkinson’s models (created through Medica, Beijing University of Chinese Medicine, Beijing 100029, China. E-mail [email protected] treatment with MPTP). Thus, FLZ possesses a strong neuro- [12] Received 2009-02-06 Accepted 2009-03-24 protective property . In addition, FLZ was also shown to

emy of Medical Sciences (Grade II, Certificate No SCXK- Jing2004-0001). They were housed in groups of 5 or 6 and had access to laboratory food and water ad libitum. Rats were maintained in a thermo-regulated environment (23±1 °C, 50%±5% humidity) in a 12-h light/dark cycle. All animal experiments were conducted in accordance with the guidelines established by the Animal Care Committee of the Peking Union Medical College and the Chinese Academy of Medical Sciences. Preparation of rat brain mitochondria Non-synaptic brain mitochondria were isolated from male Wistar rats using the published methods of Lai and Clark[18]. This preparation of nonsynaptic mitochondria is metabolically active, well coupled, and relatively free from non-mitochondrial Figure 1. Chemical structure of FLZ. contaminants[18, 19]. Mitochondrial protein concentration was determined by the Lowry method[20]. The purified mitochondria fraction was re-suspended in isolation medium reduce the impairment in learning and memory and the dam- (250 mmol/L sucrose, 0.5 mmol/L potassium EDTA, 10 age to the hippocampus induced by intracerebroventricular mmol/L Tris-HCl, pH 7.4) at 4 °C. A mitochondrial protein

(icv) injection of Aβ25–35 in mice. In SH-SY5Y cells, FLZ was concentration of 1 mg/mL was used for experiments. [14, 15] also shown to inhibit apoptosis induced by Aβ25–35 . In Determination of mitochondria swelling Changes a recent study, FLZ was effective in protecting against LPS- in mitochondrial swelling were monitored by tracking the and MPTP-induced neurotoxicity in dopaminergic neurons changes in the apparent absorbance at 520 nm on a spectro- and mice[16]. Based on these results, the present study exam- photometer over time[21]. The fresh mitochondrial protein ines whether FLZ can reduce Aβ25–35-induced dysfunction of was energized with 5 mmol/L succinate for 2 min prior to isolated rat brain mitochondria. the experiments. The reaction in 1 mL of the incubation buffer (100 mmol/L sucrose,100 mmol/L KCl, 2 mmol/L Materials and methods KH2PO4, 10 μmol/L EGTA, 5 mmol/L HEPES, pH 7.4) containing 0.15 mg of mitochondria protein was initiated

Materials Compound FLZ was kindly provided by Prof by continuous stirring at 30 °C. Aβ25–35 was incubated with Xiao-tian LIANG (Department of Pharmaceutical Chemis- mitochondria for 5 min before the addition of succinate. try, Institute of Materia Medica, Chinese Academy of Medi- Mitochondrial swelling was assayed by monitoring light scat- cal Sciences, Beijing, China). FLZ is a white powder of 99% tering at 520 nm for 30 min on a spectrophotometer. Various purity and was first dissolved in absolute ethanol and then concentrations of FLZ (1−100 μmol/L) were pre-incubated diluted with 0.9% saline to a final ethanol concentration of with mitochondria for 10 min before the addition of Aβ25–35. less than 0.5%. Aβ25–35 (Sigma, St Louis, MO, USA) was dis- The activity of α-ketoglutarate dehydrogenase (α-KGDH) solved in sterile double-distilled water at a concentration of was assayed spectrophotometrically at 25 °C by measuring 2 mg/mL, aged at 37 °C for 4 days, and subsequently stored the rate of increase in absorbance due to formation of NADH 3 [22] at -20 °C until use. Aggregation of Aβ25–35 was verified by at 340 nm (ε=6.23×10 ) . The assay mixture contained 0.2 the thioflavin-T fluorometric assay[17]. 5,5′-Dithio-bis(2- mmol/L thiamine pyrophosphate (TPP), 2 mmol/L NAD, nitrobenzoic) acid (DTNB), SDS, thiamine pyrophosphate 0.2 mmol/L CoA, 1 mmol/L MgCl2, 0.3 mmol/L D,L- (TPP), p-iodonitrotetrazolium violet (INT), D,L-dothio- dithiothreitol (DTT), 0.1% (v/v) Triton X-100, 10 mmol/L threitol (DTT), CoA, NAD and cytochrome c were pur- α-ketoglutarate, 130 mmol/L HEPES-Tris pH 7.4, and mito- chased from Sigma (St Louis, MO, USA). Trichloroacetic chondrial suspension (0.1 mg protein/mL). The reaction acid (TCA), thiobarbituric acid (TBA) and other chemical was initiated by the addition of CoA and the initial rate was reagents were of analytical grade and obtained from Beijing measured. Chemical Factory (China). The activity of pyruvate dehydrogenase (PDH) was Animals Male Wistar rats (220−250 g) were obtained assayed using the published method[24]. Briefly, the initial from the Center of Experimental Animals, Chinese Acad- buffer was made up of 50 mmol/L Tris-HCl, 0.5 mmol/L

523 Fang F et al www.nature.com/aps

EDTA, and 0.2% (v/v) Triton X-100 (pH 7.8), 2.5 mmol/L The absorbency was measured at 570 nm[27]. GSH content

NAD, 0.1 mmol/L CoA, 1 mmol/L MgCl2, 0.1 mmol/L was measured by the DTNB method. Mitochondrial suspen- oxalate, 1 mg of bovine serum albumin, 0.6 mmol/L INT, sion was incubated as described above for the mitochondrial 6.5 mmol/L phenazine methosulfate, 0.2 mmol/L TPP, swelling measurements. After incubation, mitochondria mitochondrial suspension (0.1 mg protein/mL) and 5 were isolated via centrifugation at 12 000×g for 10 min at mmol/L pyruvate to a final volume of 1 mL. After addition 4 °C. The supernatant was removed, and the protein was of 4 mmol/L TPP (50 μL) and mitochondrial suspension precipitated from the mitochondrial pellet by the addition of (50 μL, 100 μg protein), the contents were mixed, and the 20% TCA with 0.1 mol/L PBS, followed by centrifugation tubes were placed in a water bath at 37 °C for 5 min. A stable at 3000 r/min for 10 min. The supernatant, containing GSH baseline at 500 nm was then obtained for the reaction mix- from inside the mitochondria, was removed and measured ture in a double beam spectrophotometer. The reaction was using the DTNB method [28]. started by adding pyruvate to the cuvette. The absorbance Determination of cytochrome c content in mitochon- of the reaction mixture at 500 nm was recorded initially dria by Western blot The preparation of mitochondria was and then again after the reaction had proceeded for 2 min at the same as for the mitochondrial swelling experiments. At 30 °C. Units of PDH activity were calculated using a molar the end of the 30 min incubation, each mitochondrial sus- extinction coefficient of 15.4×103 for the reduced dye. pension was rapidly centrifuged at 15 000 r/min for 5 min. The activity of complex IV was measured in a final vol- The resulting pellets were resuspended in 200 μL RIPA (25 ume of 1 mL of the reaction buffer (140 mmol/L KCl, 10 mmol/L Tris-HCl, 150 mmol/L NaCl, 5 mmol/L EDTA, 5 mmol/L HEPES, pH 7.4) containing mitochondria and mmol/L EGTA, 1 mmol/L PMSF, 1% Triton X-100, 0.5% dithionite-reduced cytochrome c. The decrease in absor- Nonidet P40, 10 mg/L aprotinin and 10 mg/L leupeptin) bance of cytochrome c as it was oxidized by the enzyme was and placed on ice for 30 min. They were then centrifuged at monitored at 550 nm. The molar extinction coefficient for 12 000×g for 15 min and the supernatant was removed and cytochrome c of 19.6×103 was used[25]. stored at -70°C. Protein concentration was determined fol- ·- [20] Determination of H2O2 and O2 generation and lowing standard protocols . ·- of GSH content in mitochondria H2O2, O2 , and GSH The protein samples (100 μg) were mixed with the buf- content were measured using a spectrophotometer. Mito- fer (100 mmol/L Tris-HCl, pH=6.8, 200 mmol/L DTT, 4% chondrial protein (0.5 mg/mL) was incubated with differ- SDS, 0.2% bromophenol blue and 20% glycerol). Proteins ent concentrations of Aβ25–35 in the reaction buffer (140 were separated on 15% acrylamide gels after denaturation at mmol/L NaCl, 5.5 mmol/L glucose, 10 mmol/L potassium 100 °C for 5 min and then transferred to PVDF membranes. phosphate, pH 7.4) containing 50 μg/mL horseradish per- The membranes were incubated for 2 h at room temperature oxidase and 100 μg/mL phenol red at 37 °C for 30 min. FLZ (RT) in blocking buffer (25 mmol/L Tris-HCl, pH=7.6, 150 (1–100 μmol/L) was incubated with the mitochondria for mmol/L NaCl, and 0.01% Tween-20) containing 5% fat-

10 min at 37 °C before the addition of Aβ25–35. The incuba- free milk. Then, the blots were incubated with a primary tion was stopped by adding 10 μL 1 mol/L NaOH and then monoclonal antibody to cytochrome c (dilution of 1:1000 centrifuged at 3000 r/min for 10 min. The supernatant was in blocking buffer containing 5% fat-free milk) for 3 h at RT removed. The absorbance was measured at 595 nm with 655 with gentle shaking. After being washed three times with nm as the reference wavelength. H2O2 production was calcu- blocking buffer (5 min each), the immunoblots were incu- [26] lated using a standard curve of H2O2 . bated for 1 h at RT with anti-mouse antibody-alkaline phos- ·- O2 content was measured by NBT oxidization. Mito- phatase (1:500 dilution in blocking buffer). chondrial protein (0.5 mg/mL), was incubated with different This was followed by three washes with blocking buffer concentrations of Aβ25–35 and 20 μL 1% NBT in the reaction (5 min each). The blot was then visualized with NCBI/NBT. buffer (140 mmol/L NaCl, 5.5 mmol/L glucose, 10 mmol/L Finally, the blot was scanned and its density was analyzed potassium phosphate, pH 7.4) at 37 °C for 30 min. Various using the software[21]. concentrations of FLZ (1–100 μmol/L) were incubated Statistical analysis Data in the mitochondrial swelling with the mitochondria for 10 min at 37 °C before addition experiments are expressed as the mean±SD; other data are of Aβ25–35. The reaction was terminated in an ice bath and expressed as the mean±SEM. Statistical significance was centrifuged at 3000 r/min for 10 min. The supernatant was determined using one-way ANOVA, and P values lower than removed, and the pellet was dissolved by addition of DMSO. 0.05 were considered statistically significant.

524 www.chinaphar.com Fang F et al

Results

Effect of FLZ on Aβ25–35-induced mitochondrial swelling Usually, the opening of the mitochondrial permeability transition pore (PTP) is monitored by following the decrease in absorbance associated with mitochondrial swelling. The addition of various doses of Aβ25–35 (10 μmol/L to 30 μmol/L) caused a decrease in the absorbance in a concentration-dependent manner, which indicated mitochondrial swelling. Based on these results, we selected 20 μmol/L

Aβ25–35 as an appropriate concentration for observing the effect of FLZ on mitochondrial swelling. The results showed that 10 and 100 μmol/L FLZ significantly counteracted the decrease in absorbance induced by Aβ25–35 (Figure 2). FLZ 1 μmol/L had no effect.

Figure 2. Quantification of absorbance changes was calculated by the decrease in the absorbance at 30 min compared with the absorbance at

0 min. Mitochondria were treated with Aβ25–35 for 30 min. FLZ (1−100 µmol/L) was pre-incubated with mitochondria for 10 min before the c addition of Aβ25–35. n=3. Values are expressed as mean±SD. P<0.01 e f vs control (without Aβ25–35 and FLZ). P<0.05, P<0.01 vs Aβ25-35 (20 µmol/L) group.

Figure 3. Effect of Aβ25–35 on complex IV, α-KGDH and PDH activities of rat brain mitochondria in vitro. (A) Complex IV Effect of compound FLZ on key enzymes of mito- activity; (B) α-KGDH activity; (C) PDH activity. The isolated rat chondria intoxicated with Aβ25–35 Aβ25–35 caused a dose- brain mitochondria (100 µg/mL) were incubated with various dependent reduction in the activities of complex IV, concentrations of Aβ25–35 at 37 °C for 30 min, the three enzyme α-KGDH and PDH in isolated rat brain mitochondria. The activities were determined with different methods as described in Material and Methods. n=3−5. Data are expressed as mean±SEM. susceptibility of these enzymes to the treatment with Aβ25–35 b c P<0.05, P<0.01 vs control (without Aβ25–35). was different. To induce significant inhibition of complex IV and PDH activities, 80−100 μmol/L Aβ25–35 was necessary, whereas Aβ25–35 10 μmol/L was enough to inhibit the activity (Figure 4). of α-KGDH (Figure 3). Effect of FLZ on Aβ25–35-induced changes in H2O2, ·- Based on these results, 100 μmol/L Aβ25–35 was used to O2 production and in GSH mitochondrial content ·- study the effect of compound FLZ on complex IV and PDH Aβ25–35 increased H2O2 and O2 production in the isolated activities, and 10 μmol/L Aβ25–35 was used to test α-KGDH rat brain mitochondria in a dose dependent manner (Figure activity. The results showed that the addition of 100 μmol/ 5). The effects of 50 and 100 μmol/L Aβ25–35 were statisti-

L FLZ significantly prevented the Aβ25–35-induced reduction cally significant. Aβ25–35 50 μmol/L also decreased GSH in the activities of these three key mitochondrial enzymes content in the mitochondria (Figure 7). FLZ (100 μmol/L)

525 Fang F et al www.nature.com/aps

·- Figure 5. Effect of Aβ25–35 on H2O2 and O2 production in isolated ·- rat brain mitochondria in vitro. (A) H2O2 production. (B) O2 production. The isolated rat brain mitochondria were incubated with

different concentrations of Aβ25–35 for 30 min. n=4. mean±SEM. b P<0.05 vs control (without Aβ25–35).

induced a significant reduction in cytochrome c content in mitochondria. FLZ 100 μmol/L markedly prevented the release of cytochrome c from the mitochondria (Figure 8).

Discussion Mitochondria are essential for neuronal function and Figure 4. Effect of FLZ on complex IV, α-KGDH, and PDH activity of rat brain mitochondria inhibited by Aβ in vitro. (A) Complex morphological alterations of the mitochondria have been 25–35 [29, 30] IV activity; (B) α-KGDH activity; (C) PDH activity. Isolated rat found in AD patients . Postmortem analysis of AD brain mitochondria (100 μg/mL) were incubated with the indicated brains shows significant disturbances in mitochondrial [31, 32] concentrations of Aβ25–35 at 37 °C for 30 min. FLZ (1−100 µmol/L) energy and glucose metabolism . Impairment of mito- was pre-incubated with mitochondria for 10 min before the addition of c chondrial oxidative phosphorylation, together with a reduc- Aβ25–35. n=3. Values are expressed as mean±SEM. P<0.01 vs control e f tion in the activities of cytochrome c oxidase, pyruvate (without Aβ25–35 and FLZ); P<0.05, P<0.01 vs Aβ25–35 treated group. dehydrogenase and α-ketoglutarate dehydrogenase, seem to be responsible for the decrease in glucose metabolism [3, 33, 34] significantly protected against the Aβ25–35-induced increase in and energy production found in AD brains . Numer- ·- H2O2 and O2 production and the decrease of GSH content ous studies have shown that mitochondrial function can be in mitochondria (50 μmol/L Aβ25–35) (Figure 6, 7). disturbed by increasing secretion and intracellular accumu- [6, 7, 8, 23, 35, 36] Effect of FLZ on Aβ25–35-induced reduction in cyto- lation of Aβ or by exposure to extracellular Aβ . chrome c content in mitochondria Aβ25–35 50 μmol/L Additionally, some studies have also shown that Aβ accu-

526 www.chinaphar.com Fang F et al

Figure 7. Effect of FLZ on decrease of GSH content induced by

Aβ25–35 in rat brain mitochondria. Aβ25–35 was incubated with rat brain mitochondria for 30 min, and various concentrations of FLZ (1−100 µmol/L) were incubated with mitochondria for 10 min before the c addition of 50 μmol/L Aβ25–35. n=3. Mean±SEM. P<0.01 vs control e (without Aβ25–35 and FLZ); P<0.05 vs Aβ25–35 treated group.

thology. A reduction in the activities of these two enzymes could lead to a decrease in energy production, the accumula- tion of glutamate, and a reduction in acetylcholine synthesis. Acetylcholine and glutamate have been linked to the impairment in learning and memory observed in AD patients[39, 40]. The complex IV activity is a rate-limiting step of oxidative phosphorylation (OXPHOS) in mitochondria. The decrease in complex IV activity could result in the generation of reac- Figure 6. Effect of FLZ on H O and O ·- production in isolated rat 2 2 2 tive oxygen species (ROS) by reducing energetic synthesis brain mitochondria induced by Aβ25–35 in vitro. (A) H2O2 production. (B) O2·- production. Isolated rat brain mitochondria (500 μg protein/ and arresting the electronic transmission in the mitochon- mL) were incubated with 50 μmol/L Aβ25–35 at 37 °C for 30 mim, drial respiratory chain (MRC). Mitochondria are the larg- FLZ (1−100 µmol/L) was pre-incubated with mitochondria for 10 est source of ROS, and they also are their targets. If ROS is min before the addition of Aβ25–35. n=4. Values are expressed as c generated, it can damage mitochondrial proteins and mem- mean±SEM. P<0.01 vs control group (without Aβ25–35 and FLZ); e branes and also cause an increase in the number of mutations P<0.05 vs Aβ25–35 treated group. in the mtDNA. This inhibits the electron transport through the complexes, further stimulating mitochondrial ROS pro- mulates within the neuronal mitochondria in brains of AD duction. This results in a vicious cycle in cells[41]. [10, 37, 38] patients . Thus, there is evidence for a functional link In the present study, Aβ25–35 was shown to inhibit the between mitochondrial dysfunction and the pathogenesis of activities of three key enzymes in isolated rat brain mito- ·- AD. chondria, to increase H2O2 and O2 production, and to The main pathway for glucose oxidation in the brain decrease GSH content in the mitochondria. These results is the tricarboxylic acid (TCA) cycle, which takes place in confirmed that mitochondrial dysfunction and oxidative the mitochondria. The α-ketoglutarate dehydrogenase and stress contributed to Aβ toxicity. Pretreatment with FLZ sig- pyruvate dehydrogenase are two key enzymes of the limiting nificantly increased the activities of α-ketoglutarate dehydro- step in the TCA cycle. In addition, pyruvate dehydroge- genase, pyruvate dehydrogenase and complex IV. And FLZ ·- nase provides acetyl CoA for the synthesis of acetylcholine. also decreased H2O2 and O2 production while also increas- α-Ketoglutarate, the substrate of α-ketoglutarate dehydroge- ing GSH content in mitochondria. All these results indicate nase, is generated by glutamate dehydrogenase during the that FLZ protected against Aβ25–35-induced dysfunction and oxidative deamination of glutamate. Glutamate is an excit- oxidative stress in rat brain mitochondria. Two possible atory neurotransmitter that, in excess, can lead to neuropa- mechanisms have been proposed to explain how Aβ inhib-

527 Fang F et al www.nature.com/aps

or necrosis of cells. Cytochrome c is an essential compo- nent of the mitochondrial respiratory chain and can shuttle electrons between respiratory chain complexes III and IV because of its heme group. The release of cytochrome c leads to two potentially lethal consequences: the decrease in ATP synthesis and the overproduction of the radical superoxide anion[43]. In any case, the suppression of lipid peroxidation and inhibition of PTP opening by FLZ contribute to pre- serving mitochondrial function. In brief, compound FLZ has protective effects on the

brain mitochondrial dysfunction induced by Aβ25–35. The present results provide strong evidence to further support FLZ as a neuroprotective agent.

Acknowledgements Figure 8. Effect of FLZ on the decrease in cytochrome c content in the This project was supported by the Ministry of Science rat brain mitochondria induced by Aβ25–35. The rat brain mitochondria and Technology of China (No 2007CB507400). was previously incubated with 100 µmol/L FLZ for 10 min, and then incubated with 50 µmol/L Aβ25–35 for 30 min. The cytochrome c was determined by Western blot method as described in the Materials Author contribution and methods. n=3. Values are expressed as mean±SEM. cP < 0.01 vs f control (without Aβ25–35 and FLZ); P<0.01 vs Aβ25–35 treated group. Geng-tao LIU designed research and revised the paper; Fang FANG performed research and wrote the paper . its mitochondrial enzymes[6]. The first one is based on the References direct interaction between Aβ with these enzyme complexes in mitochondria, whereas the second one involves the pro- 1 Lin MT, Beal MF. Mitochondrial dysfunction and oxidative stress duction of reactive oxygen species that, in turn, damage pro- in neurodegenerative disease. Nature 2006; 443: 787–95. tein subunits and/or essential cofactors of mitochondria. We 2 Baloyannis SJ. Mitochondrial alterations in Alzheimer’s disease. J Alzheimers Dis 2006; 9: 119–26. had previously shown that FLZ inhibited microsomal lipid 2+ 3 Bubber P, Haroutunian V, Flsch G, Blass JP, Gibson GE. Mito peroxidation induced by Fe -cysteine. Furthermore, FLZ chondiral abnormalities in Alzheimer brain: mechanistic implica also inhibited the production of superoxide induced by LPS, tions. Ann Neurol 2005; 57: 695–703. which is probably mediated through inhibition of NADPH 4 Beal MF. Mitochondria take center stage in aging and neuro oxidase activity[16, 42]. The mechanism by which FLZ protects degeneration. Ann Neurol 2005; 58: 495–505. against the damaging effect of Aβ on mitochondria might 5 Keil U, Hauptmann S, Bonert A, Scherping I, Eckert A, Muller WE. 25–35 Mitochondrial dysfunction induced by disease relevant AbetaPP be related to its antioxidant property or to competing for the and tau protein nutations. J Alzheimers Dis 2006; 9: 139–46. binding site with the Aβ. 6 Casley CS, Canevari L, Land JM, Clark JB, Sharp MA. Beta- Furthermore, compound FLZ was shown to inhibit the amyloid inhibits integrated mitochondrial respiration and key

Aβ25-35-induced mitochondrial swelling and cytochrome c enzyme activities. J Neurochem 2002; 80: 91–100. release from mitochondria. The opening of mitochondrial 7 Casley CS, Land JM, Sharp MA, Clark JB, Duchen MR, Canevari L. Beta-amyloid fragment 25–35 causes mitochondrial dysfunction in permeability transition pores (PTP) is indicated by mito- primary cortical neurons. Neurobiol Dis 2002; 10: 258–67. chondrial swelling, which is expressed as a decrease in the 8 Abramov AY, Canevari L, Duchen MR. Beta-amyloid peptides optical density of a mitochondrial suspension. The level of induce mitochondrial dysfunction and oxidative stress in astro mitochondrial function is related to the regulation of PTP cytes and death of neurons through activation of NADPH oxidase. opening. Some anti-AD agents that prevent pathologic PTP J Neurosci 2004; 24: 565–75. opening may preserve mitochondrial function. Aβ-induced 9 Aleardi AM, Benard G, Augereau O, Malgat M, Talbot JC, Mazat JP. Gradual alteration of mitochondrial structure and function by mitochondrial dysfunction is also mediated by the opening beta-amyloids: importance of membrane viscosity changes, energy [43, 44] of mitochondrial PTP . This mitochondrial PTP open- deprivation, reactive oxygen species production and cytochrome c ing may promote cytochrome c release and cause apoptosis release. J Bioenerg Biomermbr 2005; 37: 207–25.

528 www.chinaphar.com Fang F et al

10 Lustbader JW, Cirilli M, Lin C, Xu HW, Takuma K, Wang N. spectrophotometric test in patients with infection and with phago ABAD directly links Abeta to mitochondrial toxicity in Alzheimer’s cytotic disorders. Blood 1973; 42: 121–30. disease. Science 2004; 304: 448–52. 28 Boyne AF, Ellman GL. A methodology for analysis of tissue 11 Eckert A, Keil U, Marques CA, Bonert A, Frey C, Schussel K. sulfhydryl components. Anal Biochem 1972; 46: 639–53. Mitochondrial dysfunction, apoptotic cell death and Alzheimer’s 29 Hirai K, Aliev G, Nunomura A, Fujioka H, Russell RL, Atwood CS. disease. Biochem Pharmacol 2003; 66: 1627–34. Mitochondrial abnormalities in Alzheimer’s disease. J Neurosci 12 Feng W, Wei H, Liu G. Pharmacological study of the novel 2001; 21: 3017–23. compound FLZ against experimental Parkinson’s models and its 30 Baloyannis S, Costa V, Michmizos D. Mitochondrial alterations in active mechanism. Mol Neurobiol 2005; 31: 295–300. Alzheimer’s disease. Am J Alzheimers Dis 2004; 19: 89–93. 13 Zhang D, Zhang JJ, Liu GT. The novel squamosamide derivative 31 Beal MF. Energetics in the pathogenesis of neurodegenerative (compound FLZ) attenuated 1-methyl, 4-phenyl-pyridinium diseases. Trends Neurosci 2000; 23: 298–304. ion (MPP+)-induced apoptosis and alternations of related signal 32 Blass JP, Gibson GE, Hoyer S. The role of the metabolic lesion in transduction in SH-SY5Y cells. Neuropharmacology 2007; 52: Alzheimer’s disease. J Alzheimers Dis 2002; 4: 225–32. 423–9. 33 Shoffner JM. Oxidative phosphorylation defects and Alzheimer’s 14 Fang F, Liu GT. Protective effects of compound FLZ on beta- disease. Neurogenetics 1997; 1: 13–9. amyloid peptide-(25–35)-induced mouse hippocampal injury and 34 Cottrell DA, Borthwick GM, Johnson MA, Ince PG, Turnbull DM. learning and memory impairment. Acta Pharmacol Sin 2006; 27: The role of cytochrome c oxidase deficient hippocampal neurons 651–8. in Alzheimer’s disease. Neuropathol Appl Neurobiol 2002; 28: 15 Fang F Liu GT. The novel squamosamide derivative (compound 390–6.

FLZ) attenuates Aβ25–35-induced toxicity in SH-SY5Y cells. Acta 35 Khan SM, Cassarino DS, Abramova NN, Keeney PM, Borland Pharmacol Sin 2008; 29: 152–60. MK, Trimmer PA. Alzheimer’s disease cybrids replicate beta- 16 Zhang D, Hu X, Wei SJ, Liu J, Gao H, Qian L, et al. Squamosamide amyloid abnormalities through cell death pathways. Ann Neurol derivative FLZ protects dopaminergic neurons against inflammation- 2000; 4: 148–55. mediated neurodegeneration through the inhibition of NADPH oxidase 36 Cardoso SM, Santana I, Swerdlow RH, Oliveira CR. Mitochondria activity. J Neuroinflammation 2008; 5: 21. dysfunction of Alzheimer’s disease cybrids enhances Abeta 17 Levine H. Thioflavine T interaction with synthetic Alzheimer’s toxicity. J Neurochem 2004; 89: 1417–26. disease beta-amyloid peptides: detection of amyloid aggregation in 37 Fernandez-Vizarra P, Fenandez AP, Castro-Blanco S, Serrano J, solution. Protein Sci 1993; 2: 404–10. Bentura ML, Martinez-Murillo R. Intra- and extracellular Abeta 18 Lai JC, Clark JB. Preparation of synaptic and nonsynaptic mito and PHF in clinically evaluated cases of Alzheimer’s disease. Histol chondria from mammalian brain. Methods Enzymol 1979; 55: Histopathol 2004; 19: 823–44. 51–60. 38 Teng FY, Tang BL. Widespread gamma-secretase activity in the 19 Lai JCK, Walsh JM, Dennis SC, Clark JB. Synaptic and non- cell, but do we need it at the mitochondria? Biochem Biophys Res synaptic mitochondria from rat brain: isolation and characteriza Commun 2005; 328: 1–5. tion. J Neurochem 1977; 28: 625–31. 39 Gibson GE, Blass JP, Beal MF, Victoria B. The alpha-ketoglutarate- 20 Lowry OH, Rosenbrough NJ, Farr AL, Randall RJ. Protein dehydrogenase complex: a mediator between mitochondia and measurement with the Folin phenol reagent. J Biol Chem 1951; oxidative stress in neurodegeneration. Mol Neurobiol 2005; 31: 193: 265–75. 43–63. 21 Moreira PI, Santors MS, Moreno A, Rego AC, Oliveira C. Effect of 40 Mancuso M, Siciliano G, Filosto M, Murri L. Mitochondrial amyloid β-peptide on permeability transition pore: a comparative dysfunction and Alzheimer’s disease: new development. J study. J Neurosci Res 2002; 69: 257–67. Alzheimers Dis 2006; 9: 111–7. 22 Lai JC, Cooper AJ. Brain α-ketoglutarate dehydrogenase complex: 41 Ohta S, Ohsawa I. Dysfunction of mitochondria and oxidative kinetic properties, regional distribution, and effects of inhibitors. J stress in the pathogenensis of Alzheimer’s disease: on defects in Neurochem 1986; 47: 1376–86. the cytochrome c oxidase complex and aldehyde detoxification. J 23 Canevari L, Clark JB, Bates TE. Beta-amyloid fragement 25–35 Alzheimers Dis 2006; 9: 155–66. selectivity decrease complex IV activity in isolated mitochondria. 42 Xie P, Jiao XZ, Liang XT, Feng WH, Wei HL, Liu GT. Synthesis FEBS Lett 1999; 457: 131–4. and antioxiactivity of squamosamide cyclic analogs. Acta Acad 24 Elnageh KM, Gaitonde MK. Effect of a deficiency of thiamine on Med Sin 2004; 26: 372–8. brain pyruvate dehydrogenase: enzyme assay by three different 43 Bachurin SO, Shevtsova EP, Kireeva EG, Oxenkrug GF, Sablin methods. J Neurochem 1988; 51: 1482–9. SO. Mitochondria as a target for neurotoxins and neuroprotective 25 Wharton DC, Tzagoloff A. Cytochrome oxidase from beef heart agents. Ann NY Acad Sci 2003; 993: 334–44. mitochondria. Methods Enzymol 1967; 10: 245–50. 44 Parks JK, Smith TS, Trimmer PA, Bennett JP Jr, Parker WD 26 Nathan CF, Brukner LH, Silverstein SC, Cohn ZA. Extra Jr. Neurotoxic Aβ peptides increase oxidative stress in vivo cellular cytolysis by activated macrophages and granulocytes through NMDA-receptor and nitric-oxide-synthase mechanisms, I pharmacologic triggering of effector cells and the release of and inhibit complex IV activity and induce a mitochondrial hydrogen peroxide. J Exp Med 1979; 149: 84–99. permeability transition in vitro. J Neurochem 2001; 76: 1050–6. 27 Stossel TP. Evaluation of opsonic and leukocyte function with a

529